For some time it has been understood that many oncogenic genes encode transcriptional factors. This has spurred intense interest in studying the mechanisms of transcription in order to develop better cancer therapeutics. More recently it has been realized that there may be broader health benefits related to understanding transcription in designing new pharmaceuticals that modulate endogenous cellular processes. This new frontier of drugs will rest on knowing what factors and complexes are involved in transcription and how they interact. This interaction must be understood in molecular detail to specifically regulate pathways. Currently, one of the most important and least understood aspects of eukaryotic transcription is the activation process effected by enhancer-binding proteins. The goal of this proposal is to understand this process in molecular detail. specifically, the objectives are to: 1. delimit and determine exactly what comprises an activation domain in positive activators, 2. discover how a negative regulatory protein controls this activation function, and 3. isolate and characterize the proteins and complexes that interact with the activation domains to effect transcription. These studies will be conducted in the galactose metabolism regulon of yeast. The regulation of this system is controlled by the interplay between the positive activator, GAL4, and the negative factor, GAL80. This system is offers both genetic and biochemical approaches, both of which will be extensively used. A genetic and a physical map will be created of the surfaces of GAL4 and GAL80 that interact. In connection with these efforts, new techniques will be explored for the study of protein-protein interaction. An attempt to co-crystallize the GAL80 protein with one of the GAL4-binding domains will be made. circular dichroism will be used to physically characterize a region of GAL4 defined as important for activation. In addition, an extensive mutagenic analysis will define exactly what regions of GAL4 are involved in activation. Finally, newly discovered candidate genes for encoding transcriptional factors will be analyzed further. Both biochemical and genetic strategies should uncover any other important transcription factors. It has already been demonstrated that many aspects of this system are generalizable to most if not all eukaryotic genes. It is expected that these studies will also contain broadly useful information.